p-Cymene and Rosmarinic Acid Ameliorate TNBS-Induced Intestinal Inflammation Upkeeping ZO-1 and MUC-2: Role of Antioxidant System and Immunomodulation.

p-Cymene (p-C) and rosmarinic acid (RA) are secondary metabolites that are present in medicinal herbs and Mediterranean spices that have promising anti-inflammatory properties. This study aimed to evaluate their intestinal anti-inflammatory activity in the trinitrobenzene sulphonic acid (TNBS)-induced colitis model in rats. p-C and RA (25-200 mg/kg) oral administration reduced the macroscopic lesion score, ulcerative area, intestinal weight/length ratio, and diarrheal index in TNBS-treated animals. Both compounds (200 mg/kg) decreased malondialdehyde (MDA) and myeloperoxidase (MPO), restored glutathione (GSH) levels, and enhanced fluorescence intensity of superoxide dismutase (SOD). They also decreased interleukin (IL)-1ß and tumor necrosis factor (TNF)-α, and maintained IL-10 basal levels. Furthermore, they modulated T cell populations (cluster of differentiation (CD)4+, CD8+, or CD3+CD4+CD25+) analyzed from the spleen, mesenteric lymph nodes, and colon samples, and also decreased cyclooxigenase 2 (COX-2), interferon (IFN)-γ, inducible nitric oxide synthase (iNOS), and nuclear transcription factor kappa B subunit p65 (NFκB-p65) mRNA transcription, but only p-C interfered in the suppressor of cytokine signaling 3 (SOCS3) expression in inflamed colons. An increase in gene expression and positive cells immunostained for mucin type 2 (MUC-2) and zonula occludens 1 (ZO-1) was observed. Altogether, these results indicate intestinal anti-inflammatory activity of p-C and RA involving the cytoprotection of the intestinal barrier, maintaining the mucus layer, and preserving communicating junctions, as well as through modulation of the antioxidant and immunomodulatory systems.

Weissella paramesenteroides WpK4 ameliorate the experimental amoebic colitis by increasing the expression of MUC-2 and the intestinal epithelial regeneration.

AIMS: This study evaluates the action of Weissella paramesenteroides WpK4 on amoebic colitis. METHODS AND RESULTS: Weissella paramesenteroides WpK4 was administered in Entamoeba dispar infected and noninfected mice and clinical parameters were evaluated. Following 7 days, the caeca were collected for histopathology, morphometry and immunohistochemical staining of MUC-2, CDC-47 and IgA. The treatment reduced diarrhoea and the presence of blood in the faeces and diminished the area of necrosis, also causing weight gain. Also, the addition of this bacterium enhanced the expression of the mucin (MUC-2). The reduction in necrosis and increased CDC-47 expression indicates significant epithelial regeneration. The negative correlation between CDC-47 and the necrosis area reveals that the bacterium favoured the recovery of the necrotic regions and the positive correlation found between the expression of MUC-2 and CDC-47 indicates that the epithelial regeneration also supports the synthesis of MUC-2. CONCLUSIONS: Weissella paramesenteroides WpK4 was able to increase the protection of the intestinal mucosa against experimental amoebic colitis through the increase of MUC-2 and epithelial regeneration. SIGNIFICANCE AND IMPACT OF THE STUDY: Weissella paramesenteroides WpK4 presents the potential to become a complementary tool in the treatment of amoebic colitis.

Intestinal anti-inflammatory activity of xique-xique (Pilosocereus gounellei A. Weber ex K. Schum. Bly. Ex Rowl) juice on acetic acid-induced colitis in rats.

Inflammatory bowel disease (IBD) is characterized by severe mucosal damage in the intestine and a deregulated immune response. Natural products derived from plants that are rich in bioactive compounds are used by many patients with IBD. Xique-xique (Pilosocereus gounellei) is a cactus of the Caatinga family that has been used by the local population for food and medicinal purposes. The intestinal anti-inflammatory effect of xique-xique cladode juice was evaluated in the present study. A dose of 5 mL kg-1 had a protective effect on intestinal inflammation, with an improvement in macroscopic damage, and a decrease in pro-inflammatory markers and oxidative stress, in addition to preserving the colonic tissue. Immunohistochemical analysis revealed the downregulation of IL-17, NF-κB, and iNOS, and upregulation of SOCs-1, ZO-1, and MUC-2. These protective effects could be attributed to the phenolic compounds as well as the fibers present in xique-xique juice. Further studies are needed before suggesting the use of xique-xique juice as a new alternative for treating IBD.

Entamoeba histolytica Interaction with Enteropathogenic Escherichia coli Increases Parasite Virulence and Inflammation in Amebiasis.

Epidemiological studies suggest frequent association of enteropathogenic bacteria with Entamoeba histolytica during symptomatic infection. In this study, we sought to determine if the interaction with enteropathogenic (EPEC) or nonpathogenic Escherichia coli (strain DH5α) could modify the virulence of E. histolytica to cause disease in animal models of amebiasis. In vitro studies showed a 2-fold increase in CaCo2 monolayer destruction when E. histolytica interacted with EPEC but not with E. coli DH5α for 2.5 h. This was associated with increased E. histolytica proteolytic activity as revealed by zymogram analysis and degradation of the E. histolytica CP-A1/5 (EhCP-A1/5) peptide substrate Z-Arg-Arg-pNC and EhCP4 substrate Z-Val-Val-Arg-AMC. Additionally, E. histolytica-EPEC interaction increased EhCP-A1, -A2, -A4, and -A5, Hgl, Apa, and Cox-1 mRNA expression. Despite the marked upregulation of E. histolytica virulence factors, nonsignificant macroscopic differences in amebic liver abscess development were observed at early stages in hamsters inoculated with either E. histolytica-EPEC or E. histolytica-E. coli DH5α. Histopathology of livers of E. histolytica-EPEC-inoculated animals revealed foci of acute inflammation 3 h postinoculation that progressively increased, producing large inflammatory reactions, ischemia, and necrosis with high expression of il-1ß, ifn-γ, and tnf-α proinflammatory cytokine genes compared with that in livers of E. histolytica-E. coli DH5α-inoculated animals. In closed colonic loops from mice, intense inflammation was observed with E. histolytica-EPEC manifested by downregulation of Math1 mRNA with a corresponding increase in the expression of Muc2 mucin and proinflammatory cytokine genes il-6, il-12, and mcp-1 These results demonstrate that E. histolytica/EPEC interaction enhanced the expression and production of key molecules associated with E. histolytica virulence, critical in pathogenesis and progression of disease.

Bacterial Translocation Is Linked to Increased Intestinal IFN-γ, IL-4, IL-17, and mucin-2 in Cholestatic Rats.

Background and rationale for the study. Bacterial translocation is an important triggering factor of infection and mortality in cirrhosis. In a rat model using bile duct ligation (BDL), bacterial translocation appears within 24 h after ligation. The dynamic between TH1/TH2/TH17 cytokines and the integrity of the colonic mucosa in the context of cirrhosis is little known. This study aims to determine the link between bacterial translocation and intestinal inflammation in a cholestasis model. Additionally, alterations of the colonic mucus layer and the bacterial load were also addressed. RESULTS: Bacterial translocation detected by microbiological cultures and MALDI-TOF showed that Escherichia coli predominates in mesenteric lymph nodes of BDL rats. Intestinal bacterial load analyzed by qPCR indicates a dramatic Escherichia/Shigella overgrowth at 8 and 30 days post-BDL. IFN-γ, IL-4, and IL-17 evaluated by Western blotting were increased at 8 and 30 days in the small intestine. In the colon, in contrast, only IFN-γ was significantly increased. The colonic mucus layer and mucin-2 expression determined by Alcian blue staining and immunohistochemistry surprisingly showed an increase in the mucus layer thickness related to increased mucin-2 expression during the entire process of liver damage. Hepatic enzymes, as well as collagen I, collagen III, TNF-α, and IL-6 liver gene expression were increased. In conclusion, bacterial overgrowth associated with bacterial translocation is linked to the over-expression of IFN-γ, IL-4, IL-17 and mucin-2. These molecules might facilitate the intestinal permeability through exacerbating the inflammatory process and disturbing tight junctions, leading to the perpetuation of the liver damage.

Intra-Amnionic Threonine Administered to Chicken Embryos Reduces Salmonella Enteritidis Cecal Counts and Improves Posthatch Intestinal Development.

This study assessed the effect of in ovo threonine supplementation on the response of broiler chicks challenged with Salmonella Enteritidis, considering bacterial counts in cecal contents, intestinal morphology, body weight, and weight gain. Fertilized eggs were inoculated in the amniotic fluid with saline (NT) or 3.5% threonine (T) solution at day 17.5 of incubation. At hatch, chicks were individually weighed and cloacal swabs were screened for Salmonella. At 2 days of age, half of the birds from each in ovo treatment were given either 0.5 mL of nutrient broth (sham-inoculated) or nalidixic acid-resistant Salmonella Enteritidis (SE NalR) in nutrient broth (8.3 × 107 colony forming units (CFU) SE NalR/mL). The birds were distributed using a completely randomized design with four treatments after the Salmonella challenge: no in ovo Thr supplementation and sham-inoculated in the posthatch challenge (NT-SHAM), in ovo Thr supplementation and sham-inoculated (T-SHAM), no in ovo Thr supplementation and SE NalR-challenged (NT-SE), and in ovo Thr supplementation and SE NalR-challenged (T-SE). In ovo threonine supplementation reduced Salmonella Enteritidis colonization 168-hour postinoculation and reduced the negative effects associated with Salmonella infection on intestinal morphology and performance, with results similar to those of the sham-inoculated birds. In ovo Thr supplementation increased the expression of MUC2 at hatch and the expression of MUC2 and IgA at 2 days of age and 168-hour postinoculation. Our results suggest that providing in ovo threonine promotes intestinal health in broilers challenged with Salmonella Enteritidis in the first days of life.

Temporal and spatial expression of Muc2 and Muc5ac mucins during rat respiratory and digestive tracts development.

Secreted mucins constitute a crucial part of the gel that protects respiratory and digestive epithelia, being MUC2/Muc2 the predominant gel-forming mucin of the intestine while MUC5AC/Muc5ac is one of the gel-forming mucins most expressed at the airways. In this study, we have analyzed Muc2 and Muc5ac during rat development by using immunohistochemistry, Western blotting and RT-PCR. We demonstrated that rat Muc2 was expressed in fetal intestinal goblet cells of surface epithelium of villi and developing Lieberkühn crypts. In neonates and adults, Muc2 was expressed at luminal goblet cells of small and large intestine and at gastric mucous and glandular cells. Muc5ac protein was observed in embryonic gastric and lung samples; expression increased during development and postnatal and adult life. After birth, a low reaction was detected at the tracheal surface epithelium and glands, which increased in adults.

Thyroid Transcription Factor-1 Expression in Adenocarcinomas of the Bile Duct.

BACKGROUND: The immunoreactivity of thyroid transcription factor-1 (TTF-1) is a very specific marker for lung and thyroid neoplasms; the expression of TTF-1 has also been demonstrated in extrapulmonary carcinomas. We examined the expression of TTF-1 in 15 intestinal-type adenocarcinomas of the extrahepatic bile duct. We then compared the expression to TTF-1 staining with other immunohistochemical markers including cytokeratin (CK) 7, CK20, caudal-type homeobox transcription factor 2 (CDX2), Napsin A, and MUC2. We additionally compared the clinicopathological prognostic factors with the TTF-1 expression status. RESULTS: Nuclear TTF-1 staining was detected in 2 cases (13.3%), and Napsin A was positive in the same 2 cases (13.3%). All cases were positive for CK20, CDX2, and MUC2; 5 cases were positive for CK7. There was no correlation between TTF-1 expression and the clinicopathological characteristics. CONCLUSIONS: To avoid potential pitfalls, TTF-1 should be interpreted in conjunction with the clinical setting, histology, and the results of markers such as CK7, CK20, Napsin A, and CDX2. This report is the first of TTF-1 positivity in adenocarcinomas from the extrahepatic biliary tract.

PGE(2) induces MUC2 and MUC5AC expression in human intrahepatic biliary epithelial cells via EP4/p38MAPK activation.

BACKGROUND: MUC2 and MUC5AC overproduction is considered to be associated with hepatolithiasis and related to inflammation. However, mechanisms underlying MUC upregulation under inflammatory stimulation in human intrahepatic biliary epithelial cells (HIBECs) are not completely understood. MATERIAL AND METHODS: Expression of MUC2 and MUC5AC mRNA in HIBECs was detected by real-time PCR. Expression of COX-2, EP4, and phosphorylated ERK, JNK and p38MAPK protein was detected by Western blot. Concentrations of PGE2, IL-1ß and TNF-α in cell culture supernatants were measured using the Quantikine Elisa kit. RESULTS: COX-2 expression as well as PGE2 production in HIBECs was upregulated significantly by LPS, which was completely blocked by either TLR4 antagonist or NFκB inhibitor. Selective COX-2 inhibitor suppressed LPS-induced MUC2 and MUC5AC mRNA expression remarkably. Exogenous PGE2 increased MUC2 and MUC5AC mRNA expression in a dosage-dependent manner independent of IL-1ß and TNF-α. PGE2 receptor EP4 agonist elevated MUC2 and MUC5AC expression, whereas EP4 antagonist had the opposite effect. Expression of phosphorylated p38MAPK was upregulated by exogenous PGE2, and p38MAPK inhibitor reduced MUC2 and MUC5AC expression in HIBECs. In addition, it was found that levels of PGE2, MUC2 and MUC5AC in bile samples from the hepatic ducts affected by intrahepatic stones were significantly higher than those from the unaffected hepatic ducts of patients with hepatolithiasis. CONCLUSIONS: Our findings indicate that PGE2 induces MUC2 and MUC5AC expression in HIBECs via EP4-p38MAPK signaling.

Differential expression of salivary glycoproteins in aggressive and chronic periodontitis.

OBJECTIVES: The aim of this study was to compare the pattern of secretion and the expression of mucin glycoprotein-2 (MG2) and lactoferrin in individuals with or without periodontitis. MATERIAL AND METHODS: Five individuals with aggressive periodontitis (APG), 5 with generalized chronic periodontitis (CPG) and 5 without periodontitis (CG) were enrolled after informed consent. Non-stimulated and stimulated submandibular and sublingual saliva was collected and samples analyzed by Western blot probed with specific antibodies. RESULTS: Stimulated and non-stimulated salivary flow rates did not differ among groups. Western blot analysis revealed that stimulation led to: an increase in MG2 expression in all groups, and to lactoferrin expression in APG and CPG. In non-stimulated saliva, CG exhibited the highest expression of both glycoproteins. In stimulated saliva, CG exhibited the highest expression of MG2, whereas APG the highest of lactoferrin. CONCLUSIONS: The pattern of secretion of MG2 and lactoferrin in health and disease is complex. Although the present study analyzed samples from a limited number of participants, the reduced expression of MG2 and lactoferrin in APG and CPG under non-stimulated condition, the predominant circumstance of salivary secretion during the day, suggests that these salivary constituents may play a role in the etiopathogenesis of these diseases.

Hepatocyte antigen expression in subtypes of intestinal metaplasia of the stomach.

OBJECTIVE: Recently, hepatocyte antigen (Hep) was introduced as a sensitive and reliable marker of intestinal metaplasia (IM). However, the distribution of Hep expression in subtypes of IM was not described. METHODS: We examined the expression of Hep in 58 cases of chronic gastritis associated with IM by immunohistochemical staining. Cases were classified as: 19 of IM Type I (complete) cases, 16 cases of IM Type II (incomplete) and 23 cases of IM Type III (incomplete). The distribution of Hep expression was classified into four groups according to the intensity of Hep expressing metaplastic cells: negative, low, moderate and high. We also compared expression of Hep with that of MUC-1, MUC-2 and MUC-5AC. RESULTS: Hep expression showed granular cytoplasmic staining and was specifically identified in columnar cells, but not in goblet cells. There was no significant difference between Hep expression and subtypes of IM (p > 0.005). However the difference between the distribution of Hep expression among three subtypes of IM was significant (p < 0.001). No relationship was observed among the expression of Hep, MUC-1, MUC-2 and MUC-5AC. CONCLUSION: Results of the present study revealed that the distribution of Hep expression is high in the majority of the complete type (Type I) IM cases, moderate in the majority of the incomplete Type II IM cases and low in all of the incomplete Type III IM cases and suggest that besides its role as a sensitive marker in IM, the evaluation of the distribution of Hep expression might be useful in the classification of IM.

Attenuated CagA oncoprotein in Helicobacter pylori from Amerindians in Peruvian Amazon.

Population genetic analyses of bacterial genes whose products interact with host tissues can give new understanding of infection and disease processes. Here we show that strains of the genetically diverse gastric pathogen Helicobacter pylori from Amerindians from the remote Peruvian Amazon contain novel alleles of cagA, a major virulence gene, and reveal distinctive properties of their encoded CagA proteins. CagA is injected into the gastric epithelium where it hijacks pleiotropic signaling pathways, helps Hp exploit its special gastric mucosal niche, and affects the risk that infection will result in overt gastroduodenal diseases including gastric cancer. The Amerindian CagA proteins contain unusual but functional tyrosine phosphorylation motifs and attenuated CRPIA motifs, which affect gastric epithelial proliferation, inflammation, and bacterial pathogenesis. Amerindian CagA proteins induced less production of IL-8 and cancer-associated Mucin 2 than did those of prototype Western or East Asian strains and behaved as dominant negative inhibitors of action of prototype CagA during mixed infection of Mongolian gerbils. We suggest that Amerindian cagA is of relatively low virulence, that this may have been selected in ancestral strains during infection of the people who migrated from Asia into the Americas many thousands of years ago, and that such attenuated CagA proteins could be useful therapeutically.

Lymph node involvement and not the histophatologic subtype is correlated with outcome after resection of adenocarcinoma of the ampulla of vater.

BACKGROUND: Intestinal and pancreaticobiliary types of Vater's ampulla adenocarcinoma have been considered as having different biologic behavior and prognosis. The aim of the present study was to determine the best immunohistochemical panel for tumor classification and to analyze the survival of patients having these histological types of adenocarcinoma. METHOD: Ninety-seven resected ampullary adenocarcinomas were histologically classified, and the prognosis factors were analyzed. The expression of MUC1, MUC2, MUC5AC, MUC6, CK7, CK17, CK20, CD10, and CDX2 was evaluated by using immunohistochemistry. RESULTS: Forty-three Vater's ampulla carcinomas were histologically classified as intestinal type, 47 as pancreaticobiliary, and seven as other types. The intestinal type had a significantly higher expression of MUC2 (74.4% vs. 23.4%), CK20 (76.7% vs. 29.8%), CDX2 (86% vs. 21.3%), and CD10 (81.4% vs. 51.1%), while MUC1 (53.5% vs. 82.9%) and CK7 (79.1% vs. 95.7%) were higher in pancreatobiliary adenocarcinomas. The most accurate markers for immunohistochemical classification were CDX2, MUC1, and MUC2. Survival was significantly affected by pancreaticobiliary type (p = 0.021), but only lymph node metastasis, lymphatic invasion, and stage were independent risk factors for survival in a multivariate analysis. CONCLUSION: The immunohistochemical expression of CDX2, MUC1, and MUC2 allows a reproducible classification of ampullary carcinomas. Although carcinomas of the intestinal type showed better survival in the univariate analysis, neither histological classification nor immunohistochemistry were independent predictors of poor prognosis.

Cadherin-catenin adhesion system and mucin expression: a comparison between young and older patients with gastric carcinoma.

BACKGROUND: Young patients are thought to develop gastric carcinomas with a molecular genetic profile that is distinct from that of gastric carcinomas occurring at a later age. The aim of this study was to compare the clinicopathological features and expression patterns of the markers E-cadherin and beta-catenin, and mucins (MUC1, MUC2, MUC5AC, and MUC6) in young and older patients. METHODS: The clinicopathological features and overall survival data of 62 young patients (age 40 years). A tissue microarray method and immunohistochemistry were used in order to analyze marker expression in paraffin-embedded tissue blocks obtained from both groups. RESULTS: The young group presented a higher percentage of diffuse-type tumors in comparison to the older group (P<0.01). The rates of positivity for E-cadherin and beta-catenin membranous expression patterns and mucin (MUC2, MUC5AC and MUC6) positivity were higher in the young group (P<0.01). Although young patients showed a lower frequency of alterations in marker expression and had significantly better survival rates than the older patients, neither age nor the marker expression pattern were found to be independent prognostic factors of survival. Only stage, tumor size, and tumor location persisted as prognostic factors for patients with gastric cancer. CONCLUSION: Biological markers of cellular adhesion and gastric differentiation were differently expressed in young and older patients. Our findings support the hypothesis that young patients develop carcinomas with a different genetic pathway compared to the pathway of tumors occurring at a later age, and we suggest further investigations to assess the prognostic relevance of the markers to specific subgroups.